End-to-end distance distributions and intrachain diffusion constants in unfolded polypeptide chains indicate intramolecular hydrogen bond formation
End-to-end distance distributions and intrachain diffusion constants in unfolded polypeptide chains indicate intramolecular hydrogen bond formation
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DOI：
10.1073/pnas.0604748103
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发表时间：
2006-08-15
影响因子：
11.1
通讯作者：
Kiefhaber, Thomas
中科院分区：
文献类型：
--
作者：
Moglich, Andreas;Joder, Karin;Kiefhaber, Thomas
未折叠状态的表征对于理解蛋白质折叠反应是必不可少的。我们进行了时间分辨的FRET测量，以获得有关未折叠多肽链的尺寸和内部动力学的信息。使用一种基于对从两个不同的供体-受体对获得的数据进行全局分析的方法，可以确定发色团之间的距离分布函数和扩散常数。在由16个Gly-Ser重复组成的多肽链上，FRET发色团之间的平均端到端距离从18.9埃增加到39.2埃，从0到8M GdmCl.链尺寸的增加伴随着端到端扩散常数的增加，从水中的(3.6+/-1.0)×10(-7)cm(2)S-1增加到8MGc1mCl中的(14.8+/-2.5)×10(-7)cm(2)S(-1)。这一发现表明，在水中，即使在缺乏疏水基团的非常灵活的链上也存在链内相互作用，这表明分子内形成了氢键。相互作用在变性剂结合时被破坏，这导致链灵活性增加和平均端到端距离更长。这一发现表明，在变性-未折叠蛋白质的复性过程中，多肽链的快速折叠是多肽链的固有特性，至少部分可以归因于非特异性的分子内氢键。尽管链内扩散常数降低，但在塌缩状态下，由于扩散距离较短，构象搜索加速。测量的距离分布函数和扩散常数与Szabo-SchultenSchulten理论相结合，能够重现实验确定的端到端环形成的速率常数。
Characterization of the unfolded state is essential for the understanding of the protein folding reaction. We performed timeresolved FRET measurements to gain information on the dimensions and the internal dynamics of unfolded polypepticle chains. Using an approach based on global analysis of data obtained from two different donor-acceptor pairs allowed for the determination of distance distribution functions and diffusion constants between the chromophores. Results on a polypepticle chain consisting of 16 Gly-Ser repeats between the FRET chromophores reveal an increase in the average end-to-end distance from 18.9 to 39.2 angstrom between 0 and 8 M GdmCl. The increase in chain dimensions is accompanied by an increase in the end-to-end diffusion constant from (3.6 +/- 1.0) X 10(-7) cm(2) S-1 in water to (14.8 +/- 2.5) x 10(-7) cm(2) s(-1) in 8 M Gc1mCl. This finding suggests that intrachain interactions in water exist even in very flexible chains lacking hydrophobic groups, which indicates intramolecular hydrogen bond formation. The interactions are broken upon denaturant binding, which leads to increased chain flexibility and longer average end-to-end distances. This finding implies that rapid collapse of polypepticle chains during refolding of denaturant-unfolded proteins is an intrinsic property of polypepticle chains and can, at least in part, be ascribed to nonspecific intramolecular hydrogen bonding. Despite decreased intrachain diffusion constants, the conformational search is accelerated in the collapsed state because of shorter diffusion distances. The measured distance distribution functions and diffusion constants in combination with Szabo-SchultenSchulten theory were able to reproduce experimentally determined rate constants for end-to-end loop formation.